Engineered acarid allergen and process for producing the same

ABSTRACT

The invention provides a method for producing a modified major mite allergen, obtained by altering a major allergen of house dust mites by gene engineering, which involve the steps of culturing prokaryotic or eukaryotic host cells transformed with a replication vector containing a gene encoding modified major mite allergen expressed from a promoter and collecting the expressed modified major mite allergen from the culture. The present invention provides a modified major mite allergen produced by the method, DNA molecules encoding same, and a pharmaceutical composition containing the modified major mite allergen.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a modified allergen, which is obtainedby altering a major allergen (Der f II) of house dust mites by geneengineering, and to a method for production of the modified allergen.The modified allergen obtained by the production method can be utilizedas a medicine for treating allergic diseases.

2. Description of the Related Art

It is considered that many allergic diseases are due to several kinds ofsymptoms which are developed by sensitization to the antigen causing thediseases, in which an IgE antibody specific for an allergen in bloodserum and tissue is produced, and when the antibody is exposed again tothe antigen, the antibody reacts with the antigen in each tissue.Particularly, an immediate type reaction is caused by the combination ofantigens with IgE antibodies on mast cells and basophils followed bycross-linking the IgE antibodies, and the subsequent release of severalkinds of chemical mediators from mast cells or basophils.

There is a method for controlling the binding between the antigen andthe IgE antibody as a method for treating allergic diseases. If thebinding between the antigen and the IgE antibody is controlled, thecross-linking among the IgE antibodies on mast cells or basophils, andthe release of chemical mediators are controlled to have an effect onthe treatment.

On the other hand, it appears that allergic diseases, such as bronchialasthma, childhood asthma, atopic dermatitis and the like, are mainlycaused by an allergen from mites living in house dust. Several kinds ofproteins of major mite allergens have been identified as major miteallergens (Platts-Mills et al., J. Allergy Clin. Immunol., 80, 755;1987). Furthermore, a method for mass producing a purified major miteallergen has been disclosed (Yuuki et al., Japanese J. Allergology, 39,557 (1990) and Japanese Patent Application No. 3-254683). In addition,the allergen in which a part of the above purified major mite allergenis changed, and a production method thereof has been filed (JapanesePatent Application No. 5-139793).

However, the proteins of the major allergens of mites which have beenreported and identified show problems of an allergic reaction, namelyanaphylactic shock, in hyposensitization therapy, because the activityof these allergens is high.

On the other hand, if the modified major mite allergen, which has lowerIgE-binding activity or lower allergen activity than wild-type allergensand inhibits the binding of the antigen (Der f II) and the IgE antibody,is obtained, it is possible to provide an effective medicine fortreating allergic diseases. The medicine does not show the anaphylacticshock of an allergic reaction caused by antigen administration, and itdoes not have an effect on the other parts of the immune system sincethe medicine is specific to the antigen. Modified major mite allergenshave been disclosed in Japanese Patent Application Nos. 5-139793 and5-275897. In the former application, there are problems of maintenanceand stability of immunogenicity because the molecular structure isgreatly changed by amino acid substitutions. In the latter application,it is not enough to minimize the structural change and to lower theallergen activity because a modified position of the allergen is notsatisfactorily specified, and only alanine is used as a substitute aminoacid.

SUMMARY OF THE INVENTION

The inventors of the present invention have found that, when an aminoacid of a specified part of the major mite allergen Der f II, which isalready disclosed, is replaced with other similar amino acids tominimize the structural change, it has been found that IgE-bindingactivity can be changed. It is found that there is no difference betweenthe modified major mite allergens and those of a wild-type as to theactivity that inhibits the antigen (Der f II) from binding to IgE.

An object of the present invention is to provide a method for massproducing the modified major mite allergen Der f II in which amino acidreplacements are introduced by gene engineering. Namely, the presentinvention aims to produce a material which can be utilized as a medicinefor treating allergic diseases.

The present invention involves a method in which a major allergen (Der fII) of house dust mites (Dermatophagoides farinae) is modified by geneengineering to replace amino acid residues with other amino acidresidues, prokaryotic or eukaryotic host cells, transformed with areplication vector containing a gene which encodes the modified majormite allergen, Der f II, are cultured, and the modified major miteallergen is obtained from the culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows pFLT11 which was used for amplification by a PCR method.

FIG. 2 is a graph showing the results that the absorbance of thereaction solution observed at 420 nm in Example 2, and evaluated thevalue as binding activities of modified proteins D7K (SEQ ID NO:11), D7E(SEQ ID NO:8), D7N (SEQ ID NO:10), D19E (SEQ ID NO:16), D19N (SEQ IDNO:18), R128D (SEQ ID NO:22), R128K (SEQ ID NO:24), and D129N (SEQ IDNO:26) of Der f II and human IgE. The amino acid sequences presented atthe bottom of the panels correspond to amino acid residues 1-21 of SEQID NO:2 (left panel) and amino acid residues 114-129 of SEQ ID NO:6(right panel).

DETAILED DESCRIPTION OF THE INVENTION

It has been disclosed that the major mite allergen Der f II has sixcysteine residues and has three intramolecular cross-linkages (betweenthe 8th and 119th, between 21st and 27th, and between 73rd and 78thamino acid residues) by disulfide bonds (Nishiyama et al., InternationalArchives of Allergy and Immunology, 101, 159, 1993). Further, the effectof IgE-binding activities is disclosed by preparing a modified Der f IIwhich is obtained by removing one of those disulfide bonds in themolecule by gene engineering techniques (Japanese Patent Application No.5-139793). According to the report, the disulfide bond between the 8thand 119th amino acid residues is the most important for IgE-bindingactivity.

The present inventors have repeatedly investigated and found that anarea around the disulfide bond between the 73rd cysteine and 78thcysteine has an effect on IgE-binding. Moreover, they have found that itis possible to lower the IgE-binding capability significantly byreplacing one amino acid with another amino acid having a very similarproperty. For example, the 7th aspartic acid residue can be replacedwith glutamic acid or asparagine, as well as the above-mentionedalanine. Though the 19th aspartic acid residue is replaced with glutamicacid, a change in the IgE-binding capability is not observed. However,when the aspartic acid residue is replaced with asparagine, it is foundthat the IgE-binding capability is reduced, and the capability isaffected by the electric charge at the 19th amino acid residue. Inaddition, for example, when the 9th alanine residue is replaced withleucine, the binding activity is about 30% of the original activity. Onthe other hand, when the alanine residue is replaced with proline, asshown in the present invention, it is found that the binding activity islowered to 10% or less. Among the mutants having reduced bindingactivity, effective mutants are found by an animal experiment with micethat develop mite allergy. The present invention has been attained byfinding modified allergens effective for a hyposensitization treatmentof patients with mite allergy.

The modified allergens of the present invention can be produced by anymethod suitable to the aims of the present invention, and preferably bya site-directed mutagenesis method. Although many site-directedmutagenesis methods have been established, a PCR method is readilyavailable (Ito et al., Gene, 102, 67, 1991). As an example, it is shownin the following that the 7th aspartic acid residue of the Der f IIprotein (SEQ ID NO:2) is replaced with a glutamic acid residue using theDNA chain as shown in SEQ ID NO:1.

The codon corresponding to the 7th aspartic acid residue of SEQ ID NO:8is GAT in SEQ ID NO:7. This codon is replaced with a codon correspondingto the glutamic acid residue, for example, GAG. The oligonucleotidehaving the same sequence as that of the DNA sequence near the asparticacid residue, in which only the codon (GAT) of the aspartic acid residueis replaced with a codon (GAG) of the glutamic acid residue, issynthesized (Table 1, F-D7E). This synthesis may be conducted by anywell-known method, such as conveniently performed by an automatedsynthesizer (for example, the Model 381 DNA synthesizer; manufactured byApplied Biosystems).

Any DNAfragment, including the cDNA of Der f II shown in SEQ ID NO:7,can be used as the template DNA for PCR amplification. In this case,pFLT11 (FIG. 1) was used as the template. The synthetic oligonucleotideR1 (Table 1) has the same sequence as that of the region containing aHind III recognition sequence downstream of the Der f II-coding regionon pFLT11, and PCR was conducted using the above two syntheticoligonucleotides F-D73 and R1 as primers.

After the PCR amplification, the nucleotide sequences of the resultingamplified DNA fragments can be determined by the dideoxy method (J. Mol.Biol. 162, 729-773, 1982) and the like.

Thus, modified DNA, in which the introduction of a mutation has beenconfirmed, is inserted into a cloning site of a suitable expressionvector, and a modified Der f II is expressed. In this expression, anyplasmid vector stably maintained in E. coli can be used; for example,pGEMEX1 (manufactured by Promega Company) can conveniently be used. Inthis vector, a T7 promoter is used as a high expression promoter, andthe recombinant protein is accumulated in E. coli as inclusion bodies.Many methods can be used in a chain of these operations (Maniatis et al,Molecular Cloning, Cold Spring Harbor Laboratory, 1982).

The DNA can be expressed by using a suitable vector, such as YEp13(Broach et al, Gene, 8, 121-133, 1979) in yeast. Using a yeast vectorhaving an expression cassette in combination with a modified Der f IIgene which is obtained by the method of the present invention, asuitable yeast cell can be transformed. In order to accomplish thisobject, the DNA sequence of the present invention must not be controlledby an E. coli promoter, but rather by an eukaryotic promoter, forexample, delta P8 and the like (Otake et al., Agric. Biol. Chem., 52,2753-2762, 1988).

Thus, modified plasmids were prepared by substituting amino acids, otherthan alanine, for the 7th, 9th, 19th, 128th and 129th amino acidresidues from the N terminus of the major mite allergen Der f II, andeach plasmid was used to transform E. coli and express a modified majormite allergen.

The IgE antibody binding activity of modified Der f II was thenquantitatively determined. Prior to this determination, each of themodified Der f II proteins needed to be purified as follows. Cells ofhost E. coli BL 21 carrying the above plasmid were harvested afterexpression. The cells were hypersonically crushed, and the Der f IIprotein, expressed as an inclusion body, was collected bycentrifugation. After dissolving the inclusion body with 6M urea, thesolution was dialyzed to a buffer of 20 mM Tris Hcl (pH 8.5) to removeurea and refold the protein. Then, the Der f II fraction was purified byanion exchange chromatography, i.e., the fraction including the refoldedprotein was adsorbed to a DEAE-Toyopearl (manufactured by Tosoh) columnand eluted with 80 mM NaCl to obtain the purified Der f II protein.

Using the resulting pure modified Der f II protein, the IgE-bindingactivity was quantitatively determined. To accomplish this object, aRAST-EIA kit (manufactured by Pharmacia) was conveniently used. First, adisk of filter paper activated with bromcyanide was immersed in 50 μl ofa solution of modified Der f II, which was diluted with a buffersolution of 0.1 M boric acid (pH 8.5), and was permitted to standovernight, where the protein became bound to the filter paper. Afterwashing, the filter paper was immersed in 50 μl of a serum from apatient allergic to mites, which serum was diluted four times with abuffer solution enclosed with the kit, allowed to stand for two hours at37° C., and the antigen, bound to the filter paper, was then bound tohuman anti-Der f II IgE antibodies in the serum. The reaction was thenallowed to proceed in accordance with the reaction protocol of the kit.After all reaction was finished, the absorbance of the sample at 420 nmwas used as an index of IgE-binding activity, with the results beingshown in FIG. 2. Modified proteins obtained by substituting alanine,asparagine and glutamic acid for the 7th and 19th aspartic acid residuesare noted. As to the 7th aspartic acid residue, even if it is replacedby any other amino acid, the IgE-binding ability is reduced incomparison with that of the wild-type Der f II. Accordingly, thenecessary condition for IgE-binding is that the 7th amino acid residuebe aspartic acid. On the other hand, as to the 19th aspartic acidresidue, if it is replaced by alanine and glutamine, then the bindingability is greatly reduced; but if it is replaced by glutamic acid, thebinding ability is slightly reduced. From these results, it isconsidered that, for IgE-binding, it is necessary that an acidic aminoacid be at that position. For the 9th alanine residue, the bindingactivity of the compound in which the 9th alanine residue is substitutedwith leucine is reduced to about 30% in comparison with the originalbinding activity of wild-type Der f II, and the binding activity of thecompound in which the 9th alanine residue is replaced by proline isreduced to 10% or less. As a result, it is found that the replacement ofan amino acid, which greatly contributes to the change in constitutionof the protein, reduces the binding activity.

Using the modified proteins of Der f II having a lowered IgE-bindingactivity in an animal experiment, the efficiency for hyposensitizationwas confirmed. 10 μg of Der f II and 100 μg of Freund's adjuvant wereintraperitoneally administered to male seven-week-old A/J mice tosensitize with rDer f. One mg/ml of modified proteins were intranasallyadministered to sensitized mice two times a week for three weeks. Thesame amount of a physiological salt solution was administered to thecontrols. One week after the final administration, the mice were placedinto a chamber for small animals to inhale five mg/ml of nebulized Der fII for 30 minutes. After 24 hours, the mice were killed by bleeding,followed by bronchial alveolar lavage with 1 ml of PBS. Cytospinpreparations were made from 200 μl of bronchial alveolar lavage fluid(abbreviated as BALF hereinafter), and stained with DIF-QUICK. Thesamples were observed under a microscope at 400 magnification, andleukocytes (macrophages, neutrophils, eosinophils and lymphocytes) werecounted within the range of the microscope to determine the degree ofairway inflammation. In non-sensitized mice, except for macrophages,leukocytes in BALF taken after inhalation of the antigen, are rarelyobserved. On the other hand, in immunized mice, many neutrophils andlymphocytes were observed, and the leukocyte count was about 2.5 timeshigher than that of the non-immune groups. In sensitized mice, it wasconfirmed that the airway inflammation was induced by the inhalation ofthe antigen. In contrast, in sensitized mice which were administeredeither wild-type Der f II or the substituent, the leukocyte count wassubstantially reduced, showing effective hyposensitization. Accordingly,it is proven that modified proteins of Der f II are safe and effectiveagents for treating allergic diseases, because there is littlepossibility of anaphylactic shock with the reduced IgE-binding activity.

Best Mode for Carrying Out the Invention

The following examples illustrate the present invention in details.

Example 1 Construction of an Expression Vector of a Der f II AminoAcid-substituted Mutant

A factor developing a variant in which the targeted amino acid residueof Der f II had been substituted by a different amino acid was preparedby a site-directed mutagenesis method of PCR. Before conducting the PCRmethod, oligonucleotides were synthesized as shown in Table 1.

TABLE 1 Base sequences of synthesized oligonucleotides for preparingmutants Oligonucleotides Sequences* (Mutants) NdI F-D7E(Asp7Glu)5′-GCCATATGGATCAAGTCGATGCTAAAGAGTGTGC-3′            SEQ ID NO: 27F-D7N(Asp7Asn) 5′-GCCATATGGATCAAGTCGATGCTAAAAATTGTGC-3′            SEQID NO: 28 F-D7K(Asp7Lys) 5′-GCCATATGGATCAAGTCGATGTTAAAAATGTGC-3′           SEQ ID NO: 29 R-A9P(Ala9Pro) 5′-CATTGTTGGGACAATCTT-3′           SEQ ID NO: 30 R-D19E(Asp19Glu) 5′-GTGGCAACCTTCGACCATTAC-3′           SEQ ID NO: 31 R-D19N(Asp19Asn) 5′-GTGGCAACCGTTGACCATTAC-3′           SEQ ID NO: 32 R-D19K(Asp19Lys) 5′-CCGTGGCAACCTTTGACCATTAC-3′           SEQ ID NO: 33 R-128D(Arg128Asp) 5′-CGAAGCTTAATCATCGATTTTAG-3′           SEQ ID NO: 34 R-R128K(Arg128Lys) 5′-CGAAGCTTAATCTTTGATTTT-3′           SEQ ID NO: 35 R-D129N(Asp129Asn) 5′-CGAAGCTTAATTACGGA-3′           SEQ ID NO: 36                   Hind III R15′-ATCAAGCTGGGATTTAGGTG-3′            SEQ ID NO: 37 F15′-CCCCGCGCGTTGGCCGATTC-3′            SEQ ID NO: 38 F25′-GCCCGGGAGTTCTCGATCCC-3′            SEQ ID NO: 39                   ΔBgl II F3 5′-CCGATTCATTAATGCAGCCC-3′            SEQID NO: 40 *: Boldface shows mismatching of amino acid substituents, andunderlines show recognition sites of restriction enzymes.

When the 7th Asp residue from the N terminus of Der f II was varied toGlu or Asn, a synthesized oligonucleotide was designed so as to have thesame sequence as that of wild-type Der f II, except that a codon for thetargeted amino acid to be substituted was changed to a codon for Glu orAsp, and so as to reintroduce a recognition sequence of restrictionenzyme Nde I into an upstream region of the initiation codon. PCR wascarried out using the expression vector pFLT11 (Japanese PatentApplication No. 5-139793) which includes the cDNA of the wild-type Der fII as a template. The synthesized oligonucleotide R1, having a sequencecomplementary to a region containing a Hind III recognition sequencedownstream of the site in which Der f II is cloned into pFLT11, and theabove-synthesized oligonucleotide F-D7E or F-D7N were used as primers.The PCR solution was prepared by adding: (a) one μg each of two primers,F-D7E and R1, or F-D7N and R1, to one ng of the plasmid pFLT11 as atemplate; (b) 10 μl of a 10× reaction solution attached to Taq DNApolymerase (TOYOBO), 10 μl of a 25 mM MgCl₂ solution; (c) dATP, dCTP,dGTP and dTTP so that each final concentration is 200 μM in 100 μl ofthe reaction solution; (d) distilled water to adjust the volume of thereaction solution to 100 μl; and (e) 2.5 units of the Taq DNApolymerase. After the resulting solution was left at 94° C. for oneminute to change the double-stranded DNA of the template into asingle-stranded DNA, it was left at 37° C. for two minutes to anneal theprimer into a single-stranded template, and then the solution wasreacted at 72° C. for three minutes to synthesize a complementary DNA bythe polymerase. The above steps were repeated 25 cycles to amplify theobjective DNA fragments.

After the digestion of the resulting DNA fragments by Nde I and HindIII, the fragments were inserted into the Nde I/Hind III site of plasmidpGEME1-Δ Nde (Japanese Patent Application No. 5-139793) to obtainexpression vectors carrying the DNAs (SEQ ID NO:13 and SEQ ID NO:17) ofthe mutants, pFLT11-D7E and D7N, respectively. On the other hand, whenthe 9th and 19th amino acid residues were substituted, the followingtwo-part PCR was performed. Namely, synthesized nucleotides R-A9P andR-D19N, having the same sequences as that of wild-type Der f II exceptfor the codon for the targeted amino acid residues to be changed asshown in Table 1, were prepared. The PCR reaction was conducted usingany one of those synthesized nucleotides as a primer, synthesizednucleotide F1 having the same sequence as that of the sequence about 40bps upstream from the Bgl II site of pFLT11 as another primer, andpFLT11 as a template. At the same time, a second PCR reaction wasconducted using F2, which was designed so as to be a sequence in whichit is impossible to cleave the Bgl II site on pFLT11 with the sameenzyme as a primer, the above R1 as another primer, and pFLT11 as atemplate.

Each 10 ng of these two DNA fragments obtained by PCR was added to thesolution obtained by removing the primer and the polymerase from theprevious PCR solution. The mixture was reacted at 94° C. for 10 minutes,cooled gradually to 37° C. for 30 minutes, and maintained at 37° C. for15 minutes to anneal these two fragments. Subsequently, 2.5 units of Taqpolymerase was added, and maintained at 60° C. for 30 minutes, andmaintained at 37° C. for 15 minutes to anneal these two fragments.Subsequently, 2.5 units of Taq polymerase was added and maintained at60° C. for three minutes to elongate these two DNA fragments. Then, eachof 0.5 μg of the previous two primers R1 and F1 were added, and 20cycles of PCR were repeated to amplify these fragments. By such anoperation, two kinds of fragments having the same length were obtained.One fragment had a Bgl II recognition sequence, and it was varied at thetargeted amino acid residue. The other fragment had the same amino acidsequence as that of wild type, and it was varied so as to be impossibleto be cleaved at the Bgl II recognition sequence. After these fragmentswere digested with Bgl II and Hind III, they were ligated to pGEMEX1-ΔNde I treated with Bgl II and Hind III. The only vectors that wereobtained were those in which the targeted mutations were introduced.Expression vectors pFLT11 -A9P and pFLT11 -D19N carrying cDNA (SEQ IDNO:13 and SEQ ID NO:17) of the Der f II mutants, in which the 9th and19th amino acid residues were substituted by Pro (SEQ ID NO:14) and Asn(SEQ ID NO:18), were prepared.

For the construction of the Der f II mutants R128D (SEQ ID NO:22, R128K(SEQ ID NO:24) and D129N (SEQ ID NO:26), in which the 128th and 129thamino acid residues were substituted with nucleotide sequencescorresponding to SEQ ID NOs:21, 23 and 25, respectively, the same PCRmethod as that used for substituting the 7th amino acid resideue wasused. In this case, using primers of a combination of each R-R128D,R-R128K or R-D129N and F3 having the same sequence as that of a sequenceabout 130 bps upstream from the initiation codon of Der f II, a PCRreaction was carried out under the same conditions as for D7E and thelike. The PCR products were digested with Bgl II and Hind III by thesame method as that of A9P and the like for insertion into pGEMEX1-Δ NdeI.

Example 2 Comparison of IgE-binding Abilities of the Amino AcidSubstituted Mutants of Der f II by a RAST-EIA Method

After E. coli BL21 was transformed by each expression vector of the Derf II mutants constructed in Example 1 (pFLT11-D7E, pFLT11-D7N,pFLT11-A9P, and pFLT11-D19N) and grown on an agar medium containingampicillin (1% BACTOTRYPTONE, 0.5% yeast extract, 0.5% NaCl, 1.5%BACTOAGAR (these units werew/v), 50 μg/ml ampicillin, pH 7.4), theresulting colonies were inoculated into 5 ml of an L liquid mediumcontaining ampicillin (agar was removed from the L agar mediumcontaining ampicillin). After the colonies were cultivated with shakingat 30° C. overnight, the medium was added to 500 ml of an L liquidmedium containing ampicillin, and the solution was further cultivatedwith shaking at 30° C. When the absorbancy of the solution reached 0.4,isopropyl-β-thiogalactopyranocide (IPTG) was added to 0.1 mM, and thesolution was cultivated for more five hours with shaking, with theprotein being expressed. Then, the E. coli BL21 cells, expressing eachDer f II mutant, were harvested by centrifugation, the cells were brokenwith a ultrasonic blender, and the Der f II mutants, expressed asinclusion bodies, were collected by centrifugation. The inclusion bodieswere refolded by solubilization in a buffer solution containing urea (6Murea, 100 mM Tris HCl and 10 mM ethylenediamine tetraacetic acid (EDTA),pH 7.5), and by dialysis in a Tris buffer solution (20 mM TrisHCl, pH8.5). This solution was provided on an ion exchange columnDEAE-Toyopearl (TOSOH) which was equilibrated in the above Tris buffersolution and eluted by a concentration gradient of NaCl (from 0 mM to100 mM). The fragment eluted at about 80 mM of salt concentration wassubjected to SDS-PAGE, and a purified sample was obtained as a singleband of about 14,000 of molecular weight.

Human IgE-binding activity of the wild-type Der f II and each mutant wasdetermined by using a RAST-EIA kit manufactured by Pharmacia. The methodwas performed as follows. First, a filter paper, which was activatedwith bromocyanide, was dipped in 50 μl of an antigen solution dilutedwith a buffer solution of 0.1 M boric acid (pH 8.5), and incubatedovernight at room temperature to adsorb the antigen. The antigensolution was removed, then the filter paper was washed in 500 μl of asolution of 0.1 M sodium hydrogencarbonate, dipped in 250 μl of 1 Mβ-ethanolamine (pH 9.0), and incubated for three hours at roomtemperature to prevent non-specific absorption on the paper by theblocking operation. The ethanolamine solution was removed, the paper waswashed once in 500 μl of a solution of 0.1 M sodium hydrogencarbonate,three times in 500 μl of the buffer solution of 0.1M sodium acetate (pH4.0), and twice in 500 μl of the buffer solution enclosed with the kit.50 μl of serum from an allergy patient, which was diluted 4 times withthe buffer solution enclosed with the kit, was added, and the paper inthe solution was incubated for two hours at 37° C. to combine anti-Der fII-IgE in the serum with the antigen adsorbed on the filter paper.

After the antigen-antibody reaction, the serum was removed, the filterpaper was washed three times by dipping it for ten minutes in 2.5 ml ofthe washing solution enclosed with the kit, 50 μl of an anti-humanIgE-rabbit IgG solution labeled with β-D-galactosidase in the kit wasadded, and the resulting solution was left for 16-20 ours at roomtemperature to combine the IgE adsorbed to the antigen. After theenzyme-labeled antibody solution was removed, the filter paper waswashed as described above, and 200 μl of a substrateo-nitrophenol-β-D-galactopyranoside enclosed with the kit was added toreact at 37° C. for two hours. After adding 2 ml of a solution enclosedwith the kit for stopping the enzyme reaction, the absorbance at 420 nmof the reaction solution was determined, and the value was evaluated asthe binding activity of Der f II and human IgE. The results are shown inFIG. 2. It is recognized that the IgE-binding activity of each Der f IImutant of D7E, D7N, A9P and D19N is remarkably reduced in comparisonwith the wild-type Der f II. For example, if amino acid residue Asp 7 orAsp 19, which are considered to be important for forming a human IgEepitope of Der f II based on the experiment substituting Ala therefor,was replaced with Asn which does not have a negative charge unlike Asp,it was found that the IgE-binding activity was remarkably reduced likethe Ala-substituted Der f II. As for the Asp 7 residue, even if it wasreplaced with Glu, which is an amino acid having negative charge likeAsp, the IgE-binding activity was also reduced. Moreover, bysubstituting Pro having a different side group from Ala 9, which mightbe in a recognition site of Der f II or adjacent to the site, a varianthaving strictly lower IgE-binding activity than that of the mutant A9L(the mutant in which Leu is substituted for Ala 9) was successfullymade.

Example 3 A Test for Evaluating the Effectiveness in theHyposensitization Treatment of the Amino Acid Substituted Mutant of Derf II Materials and Methods

Immunization

Ten μg of recombinant Der f II (abbreviated as rDer f II hereinafter)and 100 μl of Freund's adjuvant were intraperitoneally administeredthree times to 24 seven-week-old male A/J mice every two weeks.

Test Groups

The 24 immunized mice were divided into a control group of six mice, arDer f II administration group of six mice, a D7N administration groupof six mice, and a D19N administration group of six mice. In addition,as a reaction-negative control group, three mice, which were notimmunized, were designated as a non-immune group. Administration of rDerf II and mutants substituted by amino acid residues (D7N and D19N)

Two weeks after the final immunization, 20 μ of each of the followingwere intranasally administered twice a week for a period of three weeks:a physiological salt solution of phosphoryic acid buffer (abbreviated asPBS hereinafter) to the control group, one mg/ml of rDer f II to therDer f II administration group, one mg/ml of D7N to the D7Nadministration group, and one mg/ml of D19N to the D19N administrationgroup. The non-immune group was not treated.

A Test for Provoking Late-phase Airway Inflammation

A week after the final intranasal administration, the mice were placedinto a chamber for small animals to inhale five mg/ml of nebulized Der fII for 30 minutes. After 24 hours, the mice were killed by bleeding,followed by bronchialalveolar lavage with 1 ml of PBS. Cytospinpreparations were made from 200 μl of BALF, and stained with DIF-QUICK.The samples were observed under a microscope at 400 magnification, andleukocytes (macrophages, neutrophils, eosinophils and lymphocytes) werecounted within the range of the microscope to determine the degree ofairway inflammation.

Results

Number of Leukocytes in BALF After Inhaling rDer f II

For the non-immune group, except for macrophages, leukocytes in BALFwere rarely observed. On the other hand, for the control group ofimmunized mice, many neutrophil and lymphocytes were observed. Incomparison with non-immune groups, the leukocyte count of the controlgroup was about 2.5 times higher. For example, in the case of thecontrol group, it was confirmed that airway inflammation was provoked bythe inhalation of the antigen. However, for the groups in which rDer fII or the mutants with substituted amino acid residues, D7N and D19N,were administered, the number of neutrophils and all leukocytes weredecreased in comparison with the control group. It is clear thathyposensitization to rDer f II by the intranasal administration wasachieved. The results are shown in Table 2.

TABLE 2 Number of leukocytes (± standard error) Test groups MacrophageNeutrophil Eosinophil Lymphocyte Nonimmune 43.2 ± 5.4 0.3 ± 0.3 0.0 0.2± 0.2 Control 47.1 ± 2.4 60.0 ± 13.5 1.7 ± 1.1 2.8 ± 1.3 rDer fII 42.7 ±8.7 31.5 ± 8.9 3.7 ± 2.2 4.1 ± 1.4 D7N 46.8 ± 7.4 35.8 ± 10.5 2.9 ± 0.85.1 ± 3.0 D19N 45.9 ± 6.6 33.9 ± 7.7 3.0 ± 1.5 3.1 ± 1.7

Industrial Applicability

The modified Der f II made by gene technology according to the presentinvention can have reduced IgE-binding activity with a minimum ofmutation (one amino acid in 129 amino acids) and can be utilized forsafely treating each kind of allergy diseases caused by mites.

40 390 base pairs nucleic acid single linear cDNA unknown CDS 1..387 1GAT CAA GTC GAT GTT AAA GAT TGT GCC AAC AAT GAA ATC AAA AAA GTA 48 AspGln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 10 15ATG GTC GAT GGT TGC CAT GGT TCT GAT CCA TGC ATC ATC CAT CGT GGT 96 MetVal Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30 AAACCA TTC ACT TTG GAA GCC TTA TTC GAT GCC AAC CAA AAC ACT AAA 144 Lys ProPhe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45 ACC GCTAAA ATT GAA ATC AAA GCC AGC CTC GAT GGT CTT GAA ATT GAT 192 Thr Ala LysIle Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60 GTT CCC GGTATC GAT ACC AAT GCT TGC CAT TTT GTC AAA TGT CCA TTG 240 Val Pro Gly IleAsp Thr Asn Ala Cys His Phe Val Lys Cys Pro Leu 65 70 75 80 GTT AAA GGTCAA CAA TAT GAT ATC AAA TAT ACA TGG AAT GTG CCG AAA 288 Val Lys Gly GlnGln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys 85 90 95 ATT GCA CCA AAATCT GAA AAC GTT GTC GTT ACA GTC AAA CTT ATC GGT 336 Ile Ala Pro Lys SerGlu Asn Val Val Val Thr Val Lys Leu Ile Gly 100 105 110 GAT AAT GGT GTTTTG GCT TGC GCT ATT GCT ACC CAC GGT AAA ATC CGT 384 Asp Asn Gly Val LeuAla Cys Ala Ile Ala Thr His Gly Lys Ile Arg 115 120 125 GAT TAA 390 Asp129 amino acids amino acid linear protein unknown 2 Asp Gln Val Asp ValLys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 10 15 Met Val Asp GlyCys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30 Lys Pro Phe ThrLeu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45 Thr Ala Lys IleGlu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60 Val Pro Gly IleAsp Thr Asn Ala Cys His Phe Val Lys Cys Pro Leu 65 70 75 80 Val Lys GlyGln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys 85 90 95 Ile Ala ProLys Ser Glu Asn Val Val Val Thr Val Lys Leu Ile Gly 100 105 110 Asp AsnGly Val Leu Ala Cys Ala Ile Ala Thr His Gly Lys Ile Arg 115 120 125 Asp390 base pairs nucleic acid single linear cDNA unknown CDS 1..387 3 GATCAA GTC GAT GTT AAA GAT TGT GCC AAC AAT GAA ATC AAA AAA GTA 48 Asp GlnVal Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 130 135 140 145ATG GTC GAT GGT TGC CAT GGT TCT GAT CCA TGC ATC ATC CAT CGT GGT 96 MetVal Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 150 155 160AAA CCA TTC ACT TTG GAA GCC TTA TTC GAT GCC AAC CAA AAC ACT AAA 144 LysPro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 165 170 175ACC GCT AAA ATT GAA ATC AAA GCC AGC CTC GAT GGT CTT GAA ATT GAT 192 ThrAla Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 180 185 190GTT CCC GGT ATC GAT ACC AAT GCT TGC CAT TTT ATG AAA TGT CCA TTG 240 ValPro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 195 200 205GTT AAA GGT CAA CAA TAT GAT ATC AAA TAT ACA TGG AAT GTG CCG AAA 288 ValLys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys 210 215 220225 ATT GCA CCA AAA TCT GAA AAC GTT GTC GTT ACA GTC AAA CTT ATC GGT 336Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Ile Gly 230 235240 GAT AAT GGT GTT TTG GCT TGC GCT ATT GCT ACC CAC GGT AAA ATC CGT 384Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Gly Lys Ile Arg 245 250255 GAT TAA 390 Asp 129 amino acids amino acid linear protein unknown 4Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 1015 Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 2530 Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 4045 Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 5560 Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 65 7075 80 Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys 8590 95 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Ile Gly100 105 110 Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Gly Lys IleArg 115 120 125 Asp 390 base pairs nucleic acid single linear cDNAunknown CDS 1..387 5 GAT CAA GTC GAT GTT AAA GAT TGT GCC AAC AAT GAA ATCAAA AAA GTA 48 Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile LysLys Val 130 135 140 145 ATG GTC GAT GGT TGC CAT GGT TCT GAT CCA TGC ATCATC CAT CGT GGT 96 Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile IleHis Arg Gly 150 155 160 AAA CCA TTC ACT TTG GAA GCC TTA TTC GAT GCC AACCAA AAC ACT AAA 144 Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn GlnAsn Thr Lys 165 170 175 ACC GCT AAA ATT GAA ATC AAA GCC AGC CTC GAT GGTCTT GAA ATT GAT 192 Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly LeuGlu Ile Asp 180 185 190 GTT CCC GGT ATC GAT ACC AAT GCT TGC CAT TTT ATGAAA TGT CCA TTG 240 Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met LysCys Pro Leu 195 200 205 GTT AAA GGT CAA CAA TAT GAT GCC AAA TAT ACA TGGAAT GTG CCG AAA 288 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp AsnVal Pro Lys 210 215 220 225 ATT GCA CCA AAA TCT GAA AAC GTT GTC GTT ACAGTC AAA CTT GTT GGT 336 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr ValLys Leu Val Gly 230 235 240 GAT AAT GGT GTT TTG GCT TGC GCT ATT GCT ACCCAC GCT AAA ATC CGT 384 Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr HisAla Lys Ile Arg 245 250 255 GAT TAA 390 Asp 129 amino acids amino acidlinear protein unknown 6 Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn GluIle Lys Lys Val 1 5 10 15 Met Val Asp Gly Cys His Gly Ser Asp Pro CysIle Ile His Arg Gly 20 25 30 Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp AlaAsn Gln Asn Thr Lys 35 40 45 Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu AspGly Leu Glu Ile Asp 50 55 60 Val Pro Gly Ile Asp Thr Asn Ala Cys His PheMet Lys Cys Pro Leu 65 70 75 80 Val Lys Gly Gln Gln Tyr Asp Ala Lys TyrThr Trp Asn Val Pro Lys 85 90 95 Ile Ala Pro Lys Ser Glu Asn Val Val ValThr Val Lys Leu Val Gly 100 105 110 Asp Asn Gly Val Leu Ala Cys Ala IleAla Thr His Ala Lys Ile Arg 115 120 125 Asp 390 base pairs nucleic acidsingle linear cDNA unknown CDS 1..387 7 GAT CAA GTC GAT GTT AAA GAG TGTGCC AAC AAT GAA ATC AAA AAA GTA 48 Asp Gln Val Asp Val Lys Glu Cys AlaAsn Asn Glu Ile Lys Lys Val 130 135 140 145 ATG GTC GAT GGT TGC CAT GGTTCT GAT CCA TGC ATC ATC CAT CGT GGT 96 Met Val Asp Gly Cys His Gly SerAsp Pro Cys Ile Ile His Arg Gly 150 155 160 AAA CCA TTC ACT TTG GAA GCCTTA TTC GAT GCC AAC CAA AAC ACT AAA 144 Lys Pro Phe Thr Leu Glu Ala LeuPhe Asp Ala Asn Gln Asn Thr Lys 165 170 175 ACC GCT AAA ATT GAA ATC AAAGCC AGC CTC GAT GGT CTT GAA ATT GAT 192 Thr Ala Lys Ile Glu Ile Lys AlaSer Leu Asp Gly Leu Glu Ile Asp 180 185 190 GTT CCC GGT ATC GAT ACC AATGCT TGC CAT TTT ATG AAA TGT CCA TTG 240 Val Pro Gly Ile Asp Thr Asn AlaCys His Phe Met Lys Cys Pro Leu 195 200 205 GTT AAA GGT CAA CAA TAT GATGCC AAA TAT ACA TGG AAT GTG CCG AAA 288 Val Lys Gly Gln Gln Tyr Asp AlaLys Tyr Thr Trp Asn Val Pro Lys 210 215 220 225 ATT GCA CCA AAA TCT GAAAAC GTT GTC GTT ACA GTC AAA CTT GTT GGT 336 Ile Ala Pro Lys Ser Glu AsnVal Val Val Thr Val Lys Leu Val Gly 230 235 240 GAT AAT GGT GTT TTG GCTTGC GCT ATT GCT ACC CAC GCT AAA ATC CGT 384 Asp Asn Gly Val Leu Ala CysAla Ile Ala Thr His Ala Lys Ile Arg 245 250 255 GAT TAA 390 Asp 129amino acids amino acid linear protein unknown 8 Asp Gln Val Asp Val LysGlu Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 10 15 Met Val Asp Gly CysHis Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30 Lys Pro Phe Thr LeuGlu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45 Thr Ala Lys Ile GluIle Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60 Val Pro Gly Ile AspThr Asn Ala Cys His Phe Met Lys Cys Pro Leu 65 70 75 80 Val Lys Gly GlnGln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 85 90 95 Ile Ala Pro LysSer Glu Asn Val Val Val Thr Val Lys Leu Val Gly 100 105 110 Asp Asn GlyVal Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg 115 120 125 Asp 390base pairs nucleic acid single linear cDNA unknown CDS 1..387 9 GAT CAAGTC GAT GTT AAA AAT TGT GCC AAC AAT GAA ATC AAA AAA GTA 48 Asp Gln ValAsp Val Lys Asn Cys Ala Asn Asn Glu Ile Lys Lys Val 130 135 140 145 ATGGTC GAT GGT TGC CAT GGT TCT GAT CCA TGC ATC ATC CAT CGT GGT 96 Met ValAsp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 150 155 160 AAACCA TTC ACT TTG GAA GCC TTA TTC GAT GCC AAC CAA AAC ACT AAA 144 Lys ProPhe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 165 170 175 ACCGCT AAA ATT GAA ATC AAA GCC AGC CTC GAT GGT CTT GAA ATT GAT 192 Thr AlaLys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 180 185 190 GTTCCC GGT ATC GAT ACC AAT GCT TGC CAT TTT ATG AAA TGT CCA TTG 240 Val ProGly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 195 200 205 GTTAAA GGT CAA CAA TAT GAT GCC AAA TAT ACA TGG AAT GTG CCG AAA 288 Val LysGly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 210 215 220 225ATT GCA CCA AAA TCT GAA AAC GTT GTC GTT ACA GTC AAA CTT GTT GGT 336 IleAla Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly 230 235 240GAT AAT GGT GTT TTG GCT TGC GCT ATT GCT ACC CAC GCT AAA ATC CGT 384 AspAsn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg 245 250 255GAT TAA 390 Asp 129 amino acids amino acid linear protein unknown 10 AspGln Val Asp Val Lys Asn Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 10 15Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 65 70 7580 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 85 9095 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly 100105 110 Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg115 120 125 Asp 390 base pairs nucleic acid single linear cDNA unknownCDS 1..387 11 GAT CAA GTC GAT GTT AAA AAA TGT GCC AAC AAT GAA ATC AAAAAA GTA 48 Asp Gln Val Asp Val Lys Lys Cys Ala Asn Asn Glu Ile Lys LysVal 130 135 140 145 ATG GTC GAT GGT TGC CAT GGT TCT GAT CCA TGC ATC ATCCAT CGT GGT 96 Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile HisArg Gly 150 155 160 AAA CCA TTC ACT TTG GAA GCC TTA TTC GAT GCC AAC CAAAAC ACT AAA 144 Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln AsnThr Lys 165 170 175 ACC GCT AAA ATT GAA ATC AAA GCC AGC CTC GAT GGT CTTGAA ATT GAT 192 Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu GluIle Asp 180 185 190 GTT CCC GGT ATC GAT ACC AAT GCT TGC CAT TTT ATG AAATGT CCA TTG 240 Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys CysPro Leu 195 200 205 GTT AAA GGT CAA CAA TAT GAT GCC AAA TAT ACA TGG AATGTG CCG AAA 288 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn ValPro Lys 210 215 220 225 ATT GCA CCA AAA TCT GAA AAC GTT GTC GTT ACA GTCAAA CTT GTT GGT 336 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val LysLeu Val Gly 230 235 240 GAT AAT GGT GTT TTG GCT TGC GCT ATT GCT ACC CACGCT AAA ATC CGT 384 Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His AlaLys Ile Arg 245 250 255 GAT TAA 390 Asp 129 amino acids amino acidlinear protein unknown 12 Asp Gln Val Asp Val Lys Lys Cys Ala Asn AsnGlu Ile Lys Lys Val 1 5 10 15 Met Val Asp Gly Cys His Gly Ser Asp ProCys Ile Ile His Arg Gly 20 25 30 Lys Pro Phe Thr Leu Glu Ala Leu Phe AspAla Asn Gln Asn Thr Lys 35 40 45 Thr Ala Lys Ile Glu Ile Lys Ala Ser LeuAsp Gly Leu Glu Ile Asp 50 55 60 Val Pro Gly Ile Asp Thr Asn Ala Cys HisPhe Met Lys Cys Pro Leu 65 70 75 80 Val Lys Gly Gln Gln Tyr Asp Ala LysTyr Thr Trp Asn Val Pro Lys 85 90 95 Ile Ala Pro Lys Ser Glu Asn Val ValVal Thr Val Lys Leu Val Gly 100 105 110 Asp Asn Gly Val Leu Ala Cys AlaIle Ala Thr His Ala Lys Ile Arg 115 120 125 Asp 390 base pairs nucleicacid single linear cDNA unknown CDS 1..387 13 GAT CAA GTC GAT GTT AAAGAT TGT CCC AAC AAT GAA ATC AAA AAA GTA 48 Asp Gln Val Asp Val Lys AspCys Pro Asn Asn Glu Ile Lys Lys Val 130 135 140 145 ATG GTC GAT GGT TGCCAT GGT TCT GAT CCA TGC ATC ATC CAT CGT GGT 96 Met Val Asp Gly Cys HisGly Ser Asp Pro Cys Ile Ile His Arg Gly 150 155 160 AAA CCA TTC ACT TTGGAA GCC TTA TTC GAT GCC AAC CAA AAC ACT AAA 144 Lys Pro Phe Thr Leu GluAla Leu Phe Asp Ala Asn Gln Asn Thr Lys 165 170 175 ACC GCT AAA ATT GAAATC AAA GCC AGC CTC GAT GGT CTT GAA ATT GAT 192 Thr Ala Lys Ile Glu IleLys Ala Ser Leu Asp Gly Leu Glu Ile Asp 180 185 190 GTT CCC GGT ATC GATACC AAT GCT TGC CAT TTT ATG AAA TGT CCA TTG 240 Val Pro Gly Ile Asp ThrAsn Ala Cys His Phe Met Lys Cys Pro Leu 195 200 205 GTT AAA GGT CAA CAATAT GAT GCC AAA TAT ACA TGG AAT GTG CCG AAA 288 Val Lys Gly Gln Gln TyrAsp Ala Lys Tyr Thr Trp Asn Val Pro Lys 210 215 220 225 ATT GCA CCA AAATCT GAA AAC GTT GTC GTT ACA GTC AAA CTT GTT GGT 336 Ile Ala Pro Lys SerGlu Asn Val Val Val Thr Val Lys Leu Val Gly 230 235 240 GAT AAT GGT GTTTTG GCT TGC GCT ATT GCT ACC CAC GCT AAA ATC CGT 384 Asp Asn Gly Val LeuAla Cys Ala Ile Ala Thr His Ala Lys Ile Arg 245 250 255 GAT TAA 390 Asp129 amino acids amino acid linear protein unknown 14 Asp Gln Val Asp ValLys Asp Cys Pro Asn Asn Glu Ile Lys Lys Val 1 5 10 15 Met Val Asp GlyCys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30 Lys Pro Phe ThrLeu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45 Thr Ala Lys IleGlu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60 Val Pro Gly IleAsp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 65 70 75 80 Val Lys GlyGln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 85 90 95 Ile Ala ProLys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly 100 105 110 Asp AsnGly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg 115 120 125 Asp390 base pairs nucleic acid single linear cDNA unknown CDS 1..387 15 GATCAA GTC GAT GTT AAA GAT TGT GCC AAC AAT GAA ATC AAA AAA GTA 48 Asp GlnVal Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 130 135 140 145ATG GTC GAA GGT TGC CAT GGT TCT GAT CCA TGC ATC ATC CAT CGT GGT 96 MetVal Glu Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 150 155 160AAA CCA TTC ACT TTG GAA GCC TTA TTC GAT GCC AAC CAA AAC ACT AAA 144 LysPro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 165 170 175ACC GCT AAA ATT GAA ATC AAA GCC AGC CTC GAT GGT CTT GAA ATT GAT 192 ThrAla Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 180 185 190GTT CCC GGT ATC GAT ACC AAT GCT TGC CAT TTT ATG AAA TGT CCA TTG 240 ValPro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 195 200 205GTT AAA GGT CAA CAA TAT GAT GCC AAA TAT ACA TGG AAT GTG CCG AAA 288 ValLys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 210 215 220225 ATT GCA CCA AAA TCT GAA AAC GTT GTC GTT ACA GTC AAA CTT GTT GGT 336Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly 230 235240 GAT AAT GGT GTT TTG GCT TGC GCT ATT GCT ACC CAC GCT AAA ATC CGT 384Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg 245 250255 GAT TAA 390 Asp 129 amino acids amino acid linear protein unknown 16Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 1015 Met Val Glu Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 2530 Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 4045 Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 5560 Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 65 7075 80 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 8590 95 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly100 105 110 Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys IleArg 115 120 125 Asp 390 base pairs nucleic acid single linear cDNAunknown CDS 1..387 17 GAT CAA GTC GAT GTT AAA GAT TGT GCC AAC AAT GAAATC AAA AAA GTA 48 Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu IleLys Lys Val 130 135 140 145 ATG GTC AAC GGT TGC CAT GGT TCT GAT CCA TGCATC ATC CAT CGT GGT 96 Met Val Asn Gly Cys His Gly Ser Asp Pro Cys IleIle His Arg Gly 150 155 160 AAA CCA TTC ACT TTG GAA GCC TTA TTC GAT GCCAAC CAA AAC ACT AAA 144 Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala AsnGln Asn Thr Lys 165 170 175 ACC GCT AAA ATT GAA ATC AAA GCC AGC CTC GATGGT CTT GAA ATT GAT 192 Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp GlyLeu Glu Ile Asp 180 185 190 GTT CCC GGT ATC GAT ACC AAT GCT TGC CAT TTTATG AAA TGT CCA TTG 240 Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe MetLys Cys Pro Leu 195 200 205 GTT AAA GGT CAA CAA TAT GAT GCC AAA TAT ACATGG AAT GTG CCG AAA 288 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr TrpAsn Val Pro Lys 210 215 220 225 ATT GCA CCA AAA TCT GAA AAC GTT GTC GTTACA GTC AAA CTT GTT GGT 336 Ile Ala Pro Lys Ser Glu Asn Val Val Val ThrVal Lys Leu Val Gly 230 235 240 GAT AAT GGT GTT TTG GCT TGC GCT ATT GCTACC CAC GCT AAA ATC CGT 384 Asp Asn Gly Val Leu Ala Cys Ala Ile Ala ThrHis Ala Lys Ile Arg 245 250 255 GAT TAA 390 Asp 129 amino acids aminoacid linear protein unknown 18 Asp Gln Val Asp Val Lys Asp Cys Ala AsnAsn Glu Ile Lys Lys Val 1 5 10 15 Met Val Asn Gly Cys His Gly Ser AspPro Cys Ile Ile His Arg Gly 20 25 30 Lys Pro Phe Thr Leu Glu Ala Leu PheAsp Ala Asn Gln Asn Thr Lys 35 40 45 Thr Ala Lys Ile Glu Ile Lys Ala SerLeu Asp Gly Leu Glu Ile Asp 50 55 60 Val Pro Gly Ile Asp Thr Asn Ala CysHis Phe Met Lys Cys Pro Leu 65 70 75 80 Val Lys Gly Gln Gln Tyr Asp AlaLys Tyr Thr Trp Asn Val Pro Lys 85 90 95 Ile Ala Pro Lys Ser Glu Asn ValVal Val Thr Val Lys Leu Val Gly 100 105 110 Asp Asn Gly Val Leu Ala CysAla Ile Ala Thr His Ala Lys Ile Arg 115 120 125 Asp 390 base pairsnucleic acid single linear cDNA unknown CDS 1..387 19 GAT CAA GTC GATGTT AAA GAT TGT GCC AAC AAT GAA ATC AAA AAA GTA 48 Asp Gln Val Asp ValLys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 130 135 140 145 ATG GTC AAAGGT TGC CAT GGT TCT GAT CCA TGC ATC ATC CAT CGT GGT 96 Met Val Lys GlyCys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 150 155 160 AAA CCA TTCACT TTG GAA GCC TTA TTC GAT GCC AAC CAA AAC ACT AAA 144 Lys Pro Phe ThrLeu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 165 170 175 ACC GCT AAAATT GAA ATC AAA GCC AGC CTC GAT GGT CTT GAA ATT GAT 192 Thr Ala Lys IleGlu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 180 185 190 GTT CCC GGTATC GAT ACC AAT GCT TGC CAT TTT ATG AAA TGT CCA TTG 240 Val Pro Gly IleAsp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 195 200 205 GTT AAA GGTCAA CAA TAT GAT GCC AAA TAT ACA TGG AAT GTG CCG AAA 288 Val Lys Gly GlnGln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 210 215 220 225 ATT GCACCA AAA TCT GAA AAC GTT GTC GTT ACA GTC AAA CTT GTT GGT 336 Ile Ala ProLys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly 230 235 240 GAT AATGGT GTT TTG GCT TGC GCT ATT GCT ACC CAC GCT AAA ATC CGT 384 Asp Asn GlyVal Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg 245 250 255 GAT TAA390 Asp 129 amino acids amino acid linear protein unknown 20 Asp Gln ValAsp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 10 15 Met ValLys Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30 Lys ProPhe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45 Thr AlaLys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60 Val ProGly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 65 70 75 80 ValLys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 85 90 95 IleAla Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly 100 105 110Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg 115 120125 Asp 390 base pairs nucleic acid single linear cDNA unknown CDS1..387 21 GAT CAA GTC GAT GTT AAA GAT TGT GCC AAC AAT GAA ATC AAA AAAGTA 48 Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val130 135 140 145 ATG GTC GAT GGT TGC CAT GGT TCT GAT CCA TGC ATC ATC CATCGT GGT 96 Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His ArgGly 150 155 160 AAA CCA TTC ACT TTG GAA GCC TTA TTC GAT GCC AAC CAA AACACT AAA 144 Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn ThrLys 165 170 175 ACC GCT AAA ATT GAA ATC AAA GCC AGC CTC GAT GGT CTT GAAATT GAT 192 Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu IleAsp 180 185 190 GTT CCC GGT ATC GAT ACC AAT GCT TGC CAT TTT ATG AAA TGTCCA TTG 240 Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys ProLeu 195 200 205 GTT AAA GGT CAA CAA TAT GAT GCC AAA TAT ACA TGG AAT GTGCCG AAA 288 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val ProLys 210 215 220 225 ATT GCA CCA AAA TCT GAA AAC GTT GTC GTT ACA GTC AAACTT GTT GGT 336 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys LeuVal Gly 230 235 240 GAT AAT GGT GTT TTG GCT TGC GCT ATT GCT ACC CAC GCTAAA ATC GAT 384 Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala LysIle Asp 245 250 255 GAT TAA 390 Asp 129 amino acids amino acid linearprotein unknown 22 Asp Gln Val Asp Val Lys Asp Cys Ala Asn Asn Glu IleLys Lys Val 1 5 10 15 Met Val Asp Gly Cys His Gly Ser Asp Pro Cys IleIle His Arg Gly 20 25 30 Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala AsnGln Asn Thr Lys 35 40 45 Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp GlyLeu Glu Ile Asp 50 55 60 Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe MetLys Cys Pro Leu 65 70 75 80 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr ThrTrp Asn Val Pro Lys 85 90 95 Ile Ala Pro Lys Ser Glu Asn Val Val Val ThrVal Lys Leu Val Gly 100 105 110 Asp Asn Gly Val Leu Ala Cys Ala Ile AlaThr His Ala Lys Ile Asp 115 120 125 Asp 390 base pairs nucleic acidsingle linear cDNA unknown CDS 1..387 23 GAT CAA GTC GAT GTT AAA GAT TGTGCC AAC AAT GAA ATC AAA AAA GTA 48 Asp Gln Val Asp Val Lys Asp Cys AlaAsn Asn Glu Ile Lys Lys Val 130 135 140 145 ATG GTC GAT GGT TGC CAT GGTTCT GAT CCA TGC ATC ATC CAT CGT GGT 96 Met Val Asp Gly Cys His Gly SerAsp Pro Cys Ile Ile His Arg Gly 150 155 160 AAA CCA TTC ACT TTG GAA GCCTTA TTC GAT GCC AAC CAA AAC ACT AAA 144 Lys Pro Phe Thr Leu Glu Ala LeuPhe Asp Ala Asn Gln Asn Thr Lys 165 170 175 ACC GCT AAA ATT GAA ATC AAAGCC AGC CTC GAT GGT CTT GAA ATT GAT 192 Thr Ala Lys Ile Glu Ile Lys AlaSer Leu Asp Gly Leu Glu Ile Asp 180 185 190 GTT CCC GGT ATC GAT ACC AATGCT TGC CAT TTT ATG AAA TGT CCA TTG 240 Val Pro Gly Ile Asp Thr Asn AlaCys His Phe Met Lys Cys Pro Leu 195 200 205 GTT AAA GGT CAA CAA TAT GATGCC AAA TAT ACA TGG AAT GTG CCG AAA 288 Val Lys Gly Gln Gln Tyr Asp AlaLys Tyr Thr Trp Asn Val Pro Lys 210 215 220 225 ATT GCA CCA AAA TCT GAAAAC GTT GTC GTT ACA GTC AAA CTT GTT GGT 336 Ile Ala Pro Lys Ser Glu AsnVal Val Val Thr Val Lys Leu Val Gly 230 235 240 GAT AAT GGT GTT TTG GCTTGC GCT ATT GCT ACC CAC GCT AAA ATC AAA 384 Asp Asn Gly Val Leu Ala CysAla Ile Ala Thr His Ala Lys Ile Lys 245 250 255 GAT TAA 390 Asp 129amino acids amino acid linear protein unknown 24 Asp Gln Val Asp Val LysAsp Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 10 15 Met Val Asp Gly CysHis Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30 Lys Pro Phe Thr LeuGlu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45 Thr Ala Lys Ile GluIle Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60 Val Pro Gly Ile AspThr Asn Ala Cys His Phe Met Lys Cys Pro Leu 65 70 75 80 Val Lys Gly GlnGln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 85 90 95 Ile Ala Pro LysSer Glu Asn Val Val Val Thr Val Lys Leu Val Gly 100 105 110 Asp Asn GlyVal Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Lys 115 120 125 Asp 390base pairs nucleic acid single linear cDNA unknown CDS 1..387 25 GAT CAAGTC GAT GTT AAA GAT TGT GCC AAC AAT GAA ATC AAA AAA GTA 48 Asp Gln ValAsp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 130 135 140 145 ATGGTC GAT GGT TGC CAT GGT TCT GAT CCA TGC ATC ATC CAT CGT GGT 96 Met ValAsp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 150 155 160 AAACCA TTC ACT TTG GAA GCC TTA TTC GAT GCC AAC CAA AAC ACT AAA 144 Lys ProPhe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 165 170 175 ACCGCT AAA ATT GAA ATC AAA GCC AGC CTC GAT GGT CTT GAA ATT GAT 192 Thr AlaLys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 180 185 190 GTTCCC GGT ATC GAT ACC AAT GCT TGC CAT TTT ATG AAA TGT CCA TTG 240 Val ProGly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 195 200 205 GTTAAA GGT CAA CAA TAT GAT GCC AAA TAT ACA TGG AAT GTG CCG AAA 288 Val LysGly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 210 215 220 225ATT GCA CCA AAA TCT GAA AAC GTT GTC GTT ACA GTC AAA CTT GTT GGT 336 IleAla Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly 230 235 240GAT AAT GGT GTT TTG GCT TGC GCT ATT GCT ACC CAC GCT AAA ATC CGT 384 AspAsn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg 245 250 255AAT TAA 390 Asn 129 amino acids amino acid linear protein unknown 26 AspGln Val Asp Val Lys Asp Cys Ala Asn Asn Glu Ile Lys Lys Val 1 5 10 15Met Val Asp Gly Cys His Gly Ser Asp Pro Cys Ile Ile His Arg Gly 20 25 30Lys Pro Phe Thr Leu Glu Ala Leu Phe Asp Ala Asn Gln Asn Thr Lys 35 40 45Thr Ala Lys Ile Glu Ile Lys Ala Ser Leu Asp Gly Leu Glu Ile Asp 50 55 60Val Pro Gly Ile Asp Thr Asn Ala Cys His Phe Met Lys Cys Pro Leu 65 70 7580 Val Lys Gly Gln Gln Tyr Asp Ala Lys Tyr Thr Trp Asn Val Pro Lys 85 9095 Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Leu Val Gly 100105 110 Asp Asn Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg115 120 125 Asn 34 base pairs nucleic acid single linear cDNA unknown 27GCCATATGGA TCAAGTCGAT GCTAAAGAGT GTGC 34 34 base pairs nucleic acidsingle linear cDNA unknown 28 GCCATATGGA TCAAGTCGAT GCTAAAAATT GTGC 3434 base pairs nucleic acid single linear cDNA unknown 29 GCCATATGGATCAAGTCGAT GTTAAAAAAT GTGC 34 19 base pairs nucleic acid single linearcDNA unknown 30 CATTGTTGGG ACAATCTTT 19 21 base pairs nucleic acidsingle linear cDNA unknown 31 GTGGCAACCT TCCACCATTA C 21 21 base pairsnucleic acid single linear cDNA unknown 32 GTGGCAACCG TTGACCATTA C 21 23base pairs nucleic acid single linear cDNA unknown 33 CCGTGGCAACCTTTGACCAT TAC 23 23 base pairs nucleic acid single linear cDNA unknown34 CGAAGCTTAA TCATCGATTT TAG 23 21 base pairs nucleic acid single linearcDNA unknown 35 CGAAGCTTAA TCTTTGATTT T 21 17 base pairs nucleic acidsingle linear cDNA unknown 36 CGAAGCTTAA TTACGGA 17 20 base pairsnucleic acid single linear cDNA unknown 37 ATCAAGCTGG GATTTAGGTG 20 20base pairs nucleic acid single linear cDNA unknown 38 CCCCGCGCGTTGGCCGATTC 20 20 base pairs nucleic acid single linear cDNA unknown 39GCCCGGGAGT TCTCGATCCC 20 20 base pairs nucleic acid single linear cDNAunknown 40 CCGATTCATT AATGCAGCCC 20

What is claimed is:
 1. An isolated DNA molecule comprising a nucleotidesequence encoding a modified major mite allergen, wherein saidnucleotide sequence is modified from nucleotide sequences SEQ ID NO:1,SEQ ID NO:3, or SEQ ID NO:5, encoding wild-type major mite allergen DerfII, to substitute a codon for a different amino acid residue other thanalanine at a residue position selected from the group consisting of the7^(th), 9^(th), 19^(th), 128^(th), and 129^(th) amino acid residue ofwild-type major mite allergen Derf II.
 2. The isolated DNA moleculeaccording to claim 1, wherein said nucleotide sequence encodes amodified major mite allergen having an amino acid sequence selected fromthe group consisting of SEQ ID NOs:8, 10, 12, 14, 16, 18, 20, 22, 24 and26.
 3. The isolated DNA molecule according to claim 1, wherein saidnucleotide sequence is selected from the group consisting of SEQ IDNOs:7, 9, 11, 13, 15, 17, 19, 21, 23 and
 25. 4. A modified major miteallergen encoded by the isolated DNA molecule according to claim
 1. 5.The modified major mite allergen according to claim 4, which has reducedIgE binding activity relative to a major mite allergen of SEQ ID NO:1,SEQ ID NO:3 or SEQ ID NO:5.
 6. A pharmaceutical composition for treatingmite allergic diseases or immunizing against mite allergic diseases,comprising the modified major mite allergen according to claim 4 and apharmaceutically acceptable carrier.